This is a new application from a promising young investigator designed to study a variety of aspects of DNA methylation in the control of gene expression in early development. There are four specific aims. The first is to examine the detailed effects of methylation on the function of several viral promoters. By using a clever in vitro methylation strategy and an extrachromosomal EBV based vector system, the PI will ask whether methylation in unique sites within the tested promoters or in transcribed regions can affect expression. In the second specific aim, protein mediated DNA looping will be created in other EBV based plasmids by placing lac operator sites strategically within test plasmids and introducing these plasmids into mammalian cells which will support the replication of the EBV vectors and which constitutively express the lacI gene. By using suitably methylated substrates, the stability of methylation in looped and unlooped (by using IPTG) plasmids can be probed. The third specific aim is to investigate the role of site specific methylation of various portions of the beta globin gene region by altering the methylation patterns on P1 clones which contain the beta globin locus and introducing them into ES cells which can then be made to differentiate into cells which can express globin genes. The fourth specific aim is to try to study imprinting by isolating a P1 clone which contains the imprinted H19 and IGF2 genes and methylate them in various ways and reintroduce them into ES cells to test the effect on imprinting.
